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Biofilms and Bioshields

Existing as a bacteria is tough, especially on your own. For a pathogenic strain it's even worse, not only do they face the challenges of the environment, but the human body is full of cells whose main task within the body is to seek out and destroy them. For this reason many bacteria in the body tend to stick together to form multi-cellular-like biofilm structures which give them a better chance at surviving the body's antibacterial defenses.

Pseudomonas aeruginosa is an opportunistic human pathogen that can form biofilms and is often used to study biofilm formation. It can survive in soil, on the skin, and on a variety of surfaces, including hospital equipment such as catheters. It tends to infect humans who are already ill, and can colonise the urinary tract, kidneys and lungs, the latter making it a major secondary complication in cystic fibrosis.

One of the defenses against lung bacteria are polymorphic neutrophilic leukocytes (a type of bacteria-eating white blood cell, hereafter referred to as PMNs). These produce a variety of antibacteria chemicals, which break down invading species. However they are not able to destroy the P. aeruginosa biofilms. This isn't just because the biofilm is too complex to break into, it's due to a specific chemical; rhamnolipid. Remove the rhamnolipid from the bacteria and the PMNs can happily munch through the biofilm. This was shown with in vivo mouse studies, animals infected with bacteria unable to produce rhamnolipid contained twice as many viable PMNs in their lungs.

However rhamnolipids are not expressed all the time. They are quite potent chemicals after all, and could disrupt the surface of the cells that the P. aeruginosa are trying to grow on. They are only expressed in the presence of PMNs, as shown in the graph: (figure adapted from the reference below).The dots on the very left show wild-type P. aeruginosacells exposed to PMNs. Next, just the wild type cells, without any PMN exposure. These produce a lot less rhamnolipid. The final two results are taken from cells which are unable to produce any rhamnolipid, due to a gene knockout.

One of the most interesting things about the rhamnolipids is that they are not secreted outside the biofilm. Rather than release them into the surrounding environment the bacteria holds onto them, using them as a shield to coat the biofilm and protect it from PMN attack. Microscopy studies show that this is where the PMNs are killed as well, on the outside surface of the biofilm. This allows the action of a potentially destructive attack chemical to be carefully controlled. The rhamnolipid is only produced by the bacteria when a specific threat is present, and will then only act as a defense against cells that specifically try to attack the biofilm (microscopy figure taken from the reference below):

The biofilm is stained pink, while the surrounding PMN cells are stained blue, showing a clear dividing line between the bacteria and the surrounding attacking cells. The blue stains DNA, rather than cells, so it is the nuclei of the cells that are visible. In the top left of the picture, near the biofilm, some fuzzy blue lines can be seen, rather than the better defined blue blobs; these are DNA from PMNs that have been lysed and killed by the rhamnolipids.

The importance of this work lies in the fact that antibiotics don't really work on biofilms. Biofilms are made up of many layers of bacteria, packed deep in a peptidoglycan matrix. The antibiotic can kill off bacteria near the top of the biofilm, but can't penetrate deep enough to kill all the bacteria near the bottom. As these bacteria are exposed to a lower concentration of antibiotic (i.e not enough to kill them) they are also a lot more likely to develop resistance.

Researching interactions with the immune system therefore provides more information about new potential mechanisms for removing the bacterial infection. This is especially important for diseases like P. aeruginosa which hang around in hospitals, and are a major risk of secondary infection.

6 comments:

Love the name rhamnolipid - Ram those lipids! I wouldn't want to mess with them! Biofilms are pretty crazy, they're like a society of bacteria living in a fortress of doom. For some reason I always think of cling wrap when I picture them...

Biofilms are cool! What's not to love about bacteria doing what once was thought to be very unbacterium-like (is that even a word?)? The communication that takes place between these Pseudomonas is really fascinating. Coordinating such a simultaneous release of rhamnolipids is no small feat!

Iddo Friedberg's post on cheaters in biofilms is also a worthwile read (if you didn't already read it): http://bytesizebio.net/index.php/2009/08/25/freeloading-pays-off-but-only-up-to-a-point/

Speaking about biofilms, and in a tangent to Gram negs such as P. aeruginose, there has been some progress identifying antimicrobial agents that can act on both statonary phase and biofilm-based Gram (+)ves.

Some recent news from a conference last week in Birmingham suggests that oritavancin isn't subject to vancomycin efflux mechanism, has been more effective than vancomycin (and metronidazole, against anaerobics), as well as acting on bacteria within biofilms (and SSSi - skin and skin structure infections - treatments).

I hadn't heard of oritavancin before, I will definately have to look it up. We're on all the old-school antibiotics in my lab at the moment (vancomycin, bacitracin etc) and I haven't been keeping an eye out for new ones.

Thanks for the references! Biofilms are pretty awesome. The lab opposite mine is working on them at the moment, and I might be tempted to move in there at some point.

[I had realised that my blog posts were turning up in US time. I haven't delved into blogger enough to know how to change that yet.]

In this month's International You're probably familiar with linezolid though? In the January edition of Journal of Antimicrobial Agents there is a meta-analysis of some nine randomised controlled trials (totalling 2486 patients) assessing linezolid against vancomycin.

Will need to double check for any conflicts of interest, but the results, unsurprisingly, indicate that both have indistinguishable efficacy, though with less observed nephrotoxicity in the linezolid treatments.